1. Field of the Invention
This invention is directed to methods of modulating the activity of a p11 protein, which is shown to be involved in the regulation of tumor cell invasion, tumor growth and tumor metastasis. Disclosed are antisense polynucleotides, sense polynucleotides, siRNAs and other compositions, which modulate the activity of p11. It is disclosed herein that p11 is involved in the production of plasmin, the degradation of the extracellular matrix (“ECM”), the invasion of cells, including tumor cells, into the ECM, the growth of tumors, and the establishment of metastatic tumor foci.
2. Description of the Related Art
Anti-angiogenic Plasminogen Fragments (“AAPFs”)
Annexin II heterotetramer (“AIIt”) is a Ca2+-binding protein complex that binds t-PA, plasminogen and plasmin and stimulates both the formation and autoproteolysis of plasmin at the cell surface (Kassam et al. (1998[a]) J. Biol. Chem. 273, 4790-4799; Kassam et al. (1998[b]) Biochemistry 37, 16958-16966; Fitzpatrick et al. (2000) Biochemistry 39, 1021-1028; reviewed in Kang et al. (1999) Trends. Cardiovasc. Med. 9, 92-102). The protein consists of two copies of an annexin II 36 kDa subunit (p36) called annexin II A2 and two copies of an 11 kDa subunit (p11) called S100A10. It is known in the art that the carboxyl-terminal lysines of the p11 subunit plays a key role in plasminogen binding and activation (Kassam et al, 1998(b)).
Angiostatin was originally identified in the urine of mice bearing Lewis lung carcinoma (LLC) as a 38 kDa proteolytically-derived fragment of plasminogen which encompassed the first four kringle domains of plasminogen (Lys78-Ala440 according to SEQ ID NO:1). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases. Angiostatin was also characterized as a specific antiangiogenic protein that blocked microvascular endothelial cell proliferation but not the proliferation of nonendothelial cells (O'Reilly et al. (1994) Cold Spring Harb Symp Quant Biol 59, 471-482).
Angiostatin is a member of a family of anti-angiogenic plasminogen fragments (“AAPFs”). Physiologically relevant AAPFs include a 38 kDa AAPF isolated from the conditioned media of tumor-infiltrating macrophages (Dong et al. (1997) Cell 88, 801-810), a 43 kDa and 38 kDa AAPF identified in the conditioned media of Chinese hamster ovary and HT1080 fibrosarcoma cells and a 48 kDa AAPF present in macrophage conditioned media (Falcone et al. (1998) J. Biol. Chem. 273, 31480-31485). Other AAPFs include a 43 kDa and a 38 kDa AAPF isolated from the conditioned media of human prostrate carcinoma PC-3 cells (Gately et al. (1996) Cancer Res. 56, 4887-4890; Gately et al. (1997) PNAS USA 94, 10868-10872) and AAPFs of 66, 60 and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (Stathakis et al. (1999) J Biol Chem 274, 8910-8916). Since the carboxyl-terminus of most of these AAPFs was not determined, the exact primary sequence of most of the AAPFs is unknown.
Two distinct pathways have been identified for the formation of AAPFs. First, certain proteinases can directly cleave plasminogen into AAPFs. These proteinases include metalloelastase, gelatinase B (MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), cathepsin D and prostate-specific antigen (Patterson, B. C. and Sang, Q. A. (1997) J Biol Chem 272, 28823-28825; Cornelius et al. (1998) J. Immunol. 161, 6845-6852; Lijnen et al. (1998) Biochemistry 37, 4699-4702; Morikawa et al. (2000) J. Biol. Chem.; Heidtmann et al. (1999) Br. J. Cancer 81, 1269-1273). The source of these proteinases may be tumor-infiltrating macrophages (Dong et al., 1997) or the cancer cells themselves. For example, the conversion of plasminogen to angiostatin by macrophages is dependent on the release of metalloelastase from these cells. In comparison, Lewis lung carcinoma cells release MMP-2 which also cleaves plasminogen to angiostatin (O'Reilly et al. (1999) J Biol Chem 274, 29568-29571). Second, AAPFs are also generated by a three step mechanism which involves the conversion of plasminogen to plasmin by urokinase-type plasminogen activator (“uPA”), the autoproteolytic cleavage of plasmin and the release of the resultant plasmin fragment by cleavage of disulfide bonds. The cleavage of the plasmin disulfide bonds can be accomplished by free sulfhydryl group donors (FSD) such as glutathione or by hydroxyl ions at alkaline pH (Gately et al., 1996; Gately et al., 1997; Wu et al. (1987) PNAS USA 84, 8793-8795; Kassam et al. (2001) J Biol. Chem. 276, 8924-8933). Alternatively, the plasmin disulfide bonds can be cleaved enzymatically by a plasmin reductase such as phosphoglycerate kinase (Stathakis et al. (1997) J Biol Chem 272, 20641-20645; Lay et al. (2000) Nature 408, 869-873).
In co-pending patent application PCT/US01/44515 (published as WO0244328 A) and Kassam et al. (2001) J Biol Chem. 276, 8924-8933, which are incorporated herein by reference, it was shown by the inventor that the primary AAPF present in mouse and human blood has a molecular weight of 61 kDa. This AAPF, called A61, was produced in a cell-free system consisting of u-PA and plasminogen. A61 was shown to be a novel four-kringle containing plasminogen fragment consisting of the amino acid sequence, Lys78-Lys468 (SEQ ID NO:1) (Kassam et al., 2001). The release of A61 from plasmin required cleavage of the Lys468-Gly469 (SEQ ID NO:1) bond by plasmin autoproteolysis and also cleavage of the Cys462-Cys541 (SEQ ID NO:1) disulfide bond. Since A61 was generated in a cell-free system from plasmin at alkaline pH in the absence of sulfhydryl donors, it was concluded that cleavage of the Cys462-Cys541 disulfide was catalyzed by hydroxyl ions in vitro. In contrast, at physiological pH, it was observed that the conversion of plasminogen to A61 was very slow. These results contrasted with the observation that at physiological pH, HT1080 fibrosarcoma and bovine capillary endothelial (BCE) cells stimulated the rapid formation of A61. Heretofore, the mechanism by which these cells stimulated plasmin reduction and the release of A61 from plasmin was unclear.
Metastasis
Tumor cells are capable of escaping the constraints imposed by neighboring cells, invading the surrounding tissue and metastasizing to distant sites. This invasive property of tumor cells is dependent on the activation of proteases at the cell surface. It is generally accepted in the art that a critical factor in tumor cell invasiveness and metastasis is plasminogen activation. Plasminogen activation by cancer cells is initiated by the release of the plasminogen activator, urokinase-type plasminogen activator (“uPA”) which catalyzes the proteolytic conversion of the inactive zymogen plasminogen to the active broad-spectrum protease, plasmin, which is capable of catalyzing the degradation of proteins of the basement membrane and extracellular matrix such as laminin and fibronectin (Andreasen et al., 1997, Int J Cancer 72:1-22; Tapiovaara et al., 1996, Adv Cancer Res 69:101-133)). In addition, plasmin activates several matrix-degrading metalloproteases (MMPs) (Linjen et al., 1998, Thromb. Haemost 79:1171-1176; Mazzieri et al., 1997, EMBO J. 16:2319-2332). Thus, the generation of plasmin at the cell surface is an important event in the destruction of basement membrane and extracellular matrix that is necessary for the invasion of tumor cells into tissue (Rabbani and Mazar, 2001, Surg Oncol Clin N Am 10:393-416; Mustjoki et al., 1999, APMIS 107: 144-149).
The presence of specific receptors for both uPA and plasminogen at the cell surface is responsible for the spatial and temporal regulation of the conversion of plasminogen to plasmin (Plow et al., 1986, J Cell Biol 103:2411-2420; Stephens et al., 1989, J Cell Biol 108:1987-1995). The cell-surface receptor for uPA, urokinase-type plasminogen activator receptor (“uPAR”), acts as a scaffold for the conversion of the zymogen, pro-uPA to the catalytically active form, uPA. Subsequently, the cell surface localized uPA converts the receptor-bound plasminogen to plasmin. Binding of plasminogen to its cell surface receptors is thought to be rate-limiting for efficient activation of plasminogen by uPA (Stephens et al., 1989; Namiranian et al., 1995, Biochem J 309:977-982).
Prior to the instant invention, the identity of cellular receptors for plasminogen, which participate in uPA-dependent plasminogen activation, had not been established. It is known in the art that plasminogen binds to cells with low affinity (Kd=0.3-2 μM) and high capacity (104-107 binding sites per cell). The plasminogen binding sites on cells are heterogeneous in nature and both proteins and non-proteins such as glycosaminoglycans and gangliosides participate in plasminogen binding. A series of studies established the paradigm that only a small subset of cellular plasminogen receptors, those that possess a carboxy-terminal lysine residue, participate in cell surface plasminogen activation (reviewed in Felez, J., 1998, Fibrinolysis & Proteolysis 12:183-189). Candidate plasminogen receptors possessing carboxy-terminal lysines include p11 (Kassam et al., 1998, Biochemistry 37:16958-16966; Kassam et al., 1998, J Biol Chem 273:4790-4799; Fitzpatrick et al., 2000, Biochemistry 39:1021-1028), cytokeratin-8 (Hembrough et al., 1995, J Cell Sci 108:1071-1082; Hembrough et al., 1996, J Biol Chem 271:25684-25691; Kralovich et al., 1998, J Protein Chem 17:845-854), TIP49a (Hawley et al., 2001, J Biol Chem 276:179-186), and α-enolase (Miles at al., 1991, Biochemistry 30:1682-1691; Redlitz and Plow, 1995, Baillieres Clin Haematol 8:313-327; Pancholi, V., 2001, Cell Mol Life Sci 58:902-920).
Mainly characterized as an intracellular protein, p11 (also known in the art as, and used interchangeably with “S100A10”) is continuously expressed on the surface of different types of cells along with its binding partner, annexin II (or annexin II A2, a.k.a. p36) (Kassam et al., 1998b; Yeatman et al., 1993, Clin Exp Metastasis 11:37-44; Mai et al., 2000, J Biol Chem 275:12806-12812). Despite an abundance of in vitro kinetic data (reviewed in Kang et al., 1999, Trands Cardiovasc Med 9:92-102; Choi et al., in press, and Filipenko, 2002 18275, id), the issue of whether p11 plays an important role in the regulation of cellular plasmin generation or activity has not been addressed. Interestingly, the p11 message was observed to be upregulated in human renal cell carcinoma and gastric adenocarcinoma (Teratani, et al., 2002, Cancer Lett 175:71-77; EI-Rifai et al., 2002, Cancer Research 62:6823-6826), although a role for p11 in the development of cancer has not yet been determined or inferred in these or other publically available references to date. However, copending U.S. application Ser. No. 10/304,287 (filed on 26 Nov. 2002), which is incorporated herein by reference, teaches that p11 has plasmin reductase activity and is therefore useful in the production of antiangiogenic plasmin fragments (“AAPFs”).